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S ELECTED A ROMATIC A MINES BY G AS C HROMATOGRAPHY M ASS S PECTROMETRY : C HALLENGES OF M AINSTREAM C IGARETTE S MOKE Alexandra Martin October 3, 2013.

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Presentation on theme: "S ELECTED A ROMATIC A MINES BY G AS C HROMATOGRAPHY M ASS S PECTROMETRY : C HALLENGES OF M AINSTREAM C IGARETTE S MOKE Alexandra Martin October 3, 2013."— Presentation transcript:

1 S ELECTED A ROMATIC A MINES BY G AS C HROMATOGRAPHY M ASS S PECTROMETRY : C HALLENGES OF M AINSTREAM C IGARETTE S MOKE Alexandra Martin October 3, 2013

2 What are PAAs and why are they important? Smoke Collection 101 Brief history of PAA analysis New challenges - FDA HPHC list SPE clean-up GC-MS parameters – EI vs. NCI Validation results LOD/LOQ Accuracy Precision O UTLINE

3 W HAT A RE PAA S AND W HY A RE T HEY I MPORTANT ?

4 B ACKGROUND Tobacco smoke contains more than 4,000 chemical compounds. Characterization of this matrix has been the focus of scientists for many years. One of the goals of this work has been to identify the compounds primarily responsible for serious health effects. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans - Classification

5 PAA C LASSIFICATIONS AND R EPORTING R EQUIREMENTS Compound IARC Class Health Canada Brazil ANVISA US FDA ortho -toluidine1 X 2,6-dimethylaniline2B X ortho -anisidine2B X 1-aminonaphthalene3 XXX 2-aminonaphthalene1 XXX 3-aminobiphenylna XX 4-aminobiphenyl1 XXX

6 T HE O RIGINAL F OUR PAA S 1-aminonaphthalene2-aminonaphthalene 3-aminobiphenyl 4-aminobiphenyl

7 A DDITIONS FOR FDA HPHC L IST 2,6-dimethylaniline o -anisidine o -toluidine

8 S MOKE C OLLECTION

9 C ERULEAN 20-P ORT S MOKING M ACHINE

10

11

12 T OTAL P ARTICULATE M ATTER (TPM) FROM 5 KY 3R4F C IGARETTES

13 B RIEF H ISTORY OF PAA A NALYSIS

14 A MERICAN H EALTH F OUNDATION M ETHOD “On the Analysis of Aromatic Amines in Tobacco Smoke”. C. Patrianakos, K.D. Brunnemann and D. Hoffmann, American Health Foundation, Valhalla New York Presented at the 31 st TCRC, Greensboro, NC, October C. Patrianakos and D. Hoffmann, “Chemical Studies on Tobacco Smoke LXIV. On the Analysis of Aromatic Amines in Cigarette Smoke”, J. Anal. Toxicol., 3, (1979).

15 AHF M ETHOD (1977) Smoke Collection cigs Impingers w/ 5% HCl [Add ISTDs] Liquid:Liquid Extraction w/ Ether Dry and Concentrate Derivatize (PFPA)

16 AHF M ETHOD (1977) C ONTINUED … Florisil – 10 g Fraction (75 mL), Evaporate Florisil – 30 g Fraction 1 (500 mL) and Fraction 2 (400 mL) GC-MS or GC-ECD Analysis Wash, Dry, Evaporate Evaporate each to 2 mL

17 H EALTH C ANADA M ETHOD Health Canada Official Test Method T-102, 1999 Based upon Pieraccini et al 1992 Modifications CFP for trapping, not impinger Smaller volume solvent used Fewer evaporation steps Much better LOQs G.Pieraccini, F. Luceri and G. Moneti, “New Gas-chromatographic/Mass Spectrometric Method for the Quantitative Analysis of Primary Aromatic Amines in Main- and Side-stream Cigarette Smoke. I”, Rapid Communications in Mass Spectrometry, 6, (1992).

18 HC T-102 M ETHOD (1999) Smoke Collection - 10 cigs Extraction of CFP w/ 5% HCl [Add ISTDs] Liquid:Liquid Extraction w/ Hexane Derivatize (TMA / PFPA) GC-MS Analysis Florisil SPE (3 g)

19 N EW C HALLENGES : FDA HPHC L IST

20 G OALS OF N EW M ETHOD One method to determine all 7 PAAs. No evaporation steps. Evaluate SPE clean-up to reduce solvent use and improve reproducibility and efficiency of method. Retain the advantages of NCI (lower detection limits, improved selectivity).

21 N EW S AMPLE P REPARATION S CHEME (A RISTA M ETHOD ) Smoke Collection (CFP) Extraction of CFP w/ 5% HCl [Add ISTDs] SPE Clean-Up and Solvent Exchange (DCM) Derivatize (PFPA) GC-MS Analysis (NCI)

22 Selectivity Can selectively retain/release compounds of interest. Requires small volumes of solvent New method uses 8-10 mL vs mL with liquid:liquid. Efficiency Can process many more samples in a batch with manifolds of up to 24 ports. Effectively isolates and concentrates analytes of interest! SPE A DVANTAGES

23 W HY IS C LEAN - UP I MPORTANT ? Response Interferences can swamp the system and reduce sensitivity. Sample Throughput Dirty samples can destroy the chromatography and suppress the detector response and can increase down time due to system maintenance. Quantitation With GC/MS by NCI PAAs have simple spectra - quantitation is based on one significant daughter ion. Clean-up and separation are critical!

24 KY 3R4F E XTRACTS (N O SPE C LEAN - UP )

25 KY 3R4F E XTRACTS (C ATION E XCHANGE O NLY )

26 KY 3R4F E XTRACTS (C ATION E XCHANGE PLUS S ILICA )

27 W HICH W OULD Y OU I NJECT ?

28 C HROMATOGRAMS OF 3R4F E XTRACTS (ISO S MOKING R EGIME ) Cation plus Si SPE Cation SPE only

29 C HROMATOGRAMS OF 3R4F E XTRACTS (ISO S MOKING R EGIME ) Cation plus Si SPE Cation SPE only

30 C HROMATOGRAMS OF S TANDARD S OLUTIONS (W HY C LEAN S AMPLES ARE I MPORTANT !) 1

31 1 2

32 1 2 3

33 W HY D ERIVATIZATION ?

34 D ETECTION - EI Electron Ionization M + e - → M +  + 2e - M +  produces a characteristic fragmentation pattern or a “mass spectrum” specific to each compound.

35 D ETECTION – NCI Chemical Ionization (methane) CH 4 + e - → CH 4 +  + 2e - The electrons have much lower energies. Most compounds cannot form stable negative ions but react with the CH 4 +  (PCI). If a compound can capture and hold an extra electron and retain a negative charge, it can be observed in NCI mode. MF + e - → MF - → [M-H] -  + HF

36 EI VS. NCI S PECTRA FOR O -T OLUIDINE

37 EI VS. NCI S PECTRA FOR 4- A MINOBIPHENYL

38 C HROMATOGRAM OF L OW S TANDARD (EI VS. NCI) EI

39 C HROMATOGRAM OF L OW S TANDARD (EI VS. NCI) EI NCI

40 C HROMATOGRAMS OF A C IGAR E XTRACT (EI VS. NCI) 1-aminonaphthalene 2-aminonaphthalene 3- aminobiphenyl 4-aminobiphenyl o -toluidine o - anisidine 2,6-DMA o -toluidine 2,6-DMA o - anisidine 1-aminonaphthalene 2-aminonaphthalene 3- aminobiphenyl 4-aminobiphenyl EI NCI

41 M ETHOD V ALIDATION

42 V ALIDATION P ARAMETERS o - toluidine 2,6- DMA o - anisidine 3-Day Precision (n=20) 3.7%6.0%5.2% Accuracy (n=49)99.6%95.4%95.1% LOD (ng/mL) LOQ (ng/mL) Linearity (ng/mL)

43 V ALIDATION P ARAMETERS 1-amn2-amn3-amb4-amb 3-Day Precision (n=20) 4.3%5.2%5.5%5.0% Accuracy (n=49)95.8%100.8%94.3%91.5% LOD (ng/mL) LOQ (ng/mL) Linearity (ng/mL)

44 M ETHOD C OMPARISON (W E ’ VE C OME A L ONG W AY, B ABY ) AHF (1977) HC T- 102 (1999) Arista (2012) No. of Cigarettes Volume Organic Solvent (mL) > 3000~ 3006 – 8 No. of SPE Steps212 No. of Evaporation Steps42None Final Volume (mL)215 ~ 4-AMB LOQ (ng/mL)

45 LCMSMS J. Schubert, O. Kappenstein, A. Luch and T. G. Schulz, “Analysis of primary aromatic amines in mainstream waterpipe smoke using liquid chromatography-electrospray ionization tandem mass spectrometry”, J. Chrom. A, 1218, (2011).

46 S UMMARY Achieving lower and lower detection limits in complicated matrix requires both improving clean-up (SPE) and selectivity of the instrument (NCI). The method presented here demonstrates excellent detection limits, selectivity, accuracy and precision. It is robust, efficient and fit-for-purpose, accommodating the regulatory requirements of Health Canada, ANVISA and the US FDA.

47 T HANK Y OU !


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